Proteins move across lipid bilayers through membrane-embedded transporters, called translocase channels. These types of transporters are critical to the formation of membrane-encapsulated organelles and protein secretion. Translocase channels are also used by pathogenic bacteria to deliver cytotoxic proteins and peptides into eukaryotic host cells. The molecular basis of protein translocation, however, is poorly understood. Several models have been invoked to describe how a chemical or transmembrane potential gradient energy source can be transduced into a directed mechanical force that promotes unfolding and translocation. On one hand, an extended-chain model considers the channel to be a static structure through which the translocating peptide translocates as an extended chain with little helical structure. In this model, net movement of the peptide is explained by a proton-powered Brownian ratchet. On the other hand, the helix-compaction model hypothesizes that the translocating chain contracts from an extended-chain conformation to a helical one. This conformational transition is coordinated by an allosteric conformational change in the channel that can accommodate helical structure. The allosteric transition may be triggered by proton binding and dynamic transitions in the polypeptide clamp active sites located along the length of the transporter. Using anthrax toxin as model system, this proposal seeks to distinguish these two models using high-resolution electron microscopy (EM), single-channel electrophysiology, and cross-linking mass spectrometry (MS). The bacterium, Bacillus anthracis secretes the three-protein toxin, anthrax toxin, which is composed of protective antigen (PA), lethal factor (LF), and edema factor (EF). PA is the translocase channel that delivers the enzymatic factors, LF and EF, into the host cytosol. PA first co-assembles with LF and EF to form an oligomeric toxin complex that is endocytosed. Within the endosomal membrane, PA inserts and forms a narrow aqueous passageway through which LF and EF unfold and translocate through to reach the other side. Polypeptide clamp sites within the PA channel have been found to be dynamic active sites, which can bind and release the translocating chain of EF and LF. A recent high- resolution electron microscopy structure has revealed a narrow configuration of the central phenylalanine clamp (? clamp) site. However, this structure does not account for an alternate configuration of the clamp anticipated from single-channel electrophysiology and genetic co-variation of putative contacting residues in the ?-clamp loop. These configurations will be analyzed structurally and thermodynamically by mutational and pH-dependent studies of the channel. To gain insight on the structural configurations of the translocating LF inside the channel, conformationally locked substrates will be trapped for detailed structural analysis. Relevance: Insight on the mechanism of protein translocation is of translational relevance to the development of novel methods to neutralize the toxin and also to advancing technologies, which exploit toxins as nanopore biosensors and versatile delivery vehicles for heterologous antigens and cytotoxins into cells.